1. Field of the Invention
This invention relates to new inhibitors of trypsin-like serine proteases, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds as thrombin inhibitors and anticoagulants and as antiinflammatory inhibitors.
2. Related Art
Proteases are enzymes that cleave proteins at single, specific peptide bonds. Proteases can be classified into four generic classes: serine, thiol or cysteinyl, acid or aspartyl, and metalloproteases (Cuypers et al., J. Biol. Chem. 257:7086 (1982)). Proteases are essential to a variety of biological activities, such as digestion, formation and dissolution of blood clots, reproduction and the immune reaction to foreign cells and organisms. Aberrant proteolysis is associated with a number of disease states in man and other mammals. The human neutrophil proteases, elastase and cathepsin G, have been implicated as contributing to disease states marked by tissue destruction. These disease states include emphysema, rheumatoid arthritis, comeal ulcers and glomerular nephritis. (Barret, in Enzyme Inhibitors as Drugs, Sandler, ed., University Park Press, Baltimore, (1980)). Additional proteases such as plasmin, C-1 esterase, C-3 convertase, urokinase, plasminogen activator, acrosin, and kallikreins play key roles in normal biological functions of mammals. In many instances, it is beneficial to disrupt the function of one or more proteolytic enzymes in the course of therapeutically treating a mammal.
Serine proteases include such enzymes as elastase (human leukocyte), cathepsin G, plasmin, C-1 esterase, C-3 convertase, urokinase, plasminogen activator, acrosin, chymotrypsin, trypsin, thrombin, factor Xa and kallikreins.
Human leukocyte elastase is released by polymorphonuclear leukocytes at sites of inflammation and thus is a contributing cause for a number of disease states. Cathepsin G is another human neutrophil serine protease. Compounds with the ability to inhibit the activity of these enzymes are expected to have an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, and in the treatment of emphysema. Chymotrypsin and trypsin are digestive enzymes. Inhibitors of these enzymes are useful in treating pancreatitis. Inhibitors of urokinase and plasminogen activator are useful in treating excessive cell growth disease states, such as benign prostatic hypertrophy, prostatic carcinoma and psoriasis.
Inhibitors of thrombin based on the amino acid sequence around the cleavage site for the fibrinogen Axcex1 chain were first reported by Blowback et al, J. Clin. Lab. Invest. 24, suppl.:107, 59 (1969), who suggested the sequence Phe-Val-Arg (P9-P2-P1, herein referred to as the P3-P2-P1 sequence) to be the best inhibitor.
U.S. Pat. No. 4,346,078 discloses the thrombin inhibitor H-DPhe-Pro-Agm, a dipeptidyl derivative with an aminoalkyl guanidine in the P1-position.
A number of dipeptidyl analogs of H-DPhe-Pro-Arg, and their use as thrombin inhibitors are described in U.S. Pat. Nos. 5,602,253, 5,614,499 and PCT Published Appl. No. WO 97/46577.
Inhibitors of thrombin based on peptide derivatives with a cyclic aminoalkyl guanidine, e.g. 3-aminomethyl-1-amidinopiperidine, in the P1-position have been disclosed in EP-A2-0,468,231.
Inhibitors of kininogenases that comprise a dipeptide linked to aminoalkyl guanidines are disclosed in PCT Published Appl. No. WO 95/07291.
PCT Published Appl. No. WO 92/04371 describing kininogenase inhibitors, e.g. kallikrein inhibitors based on derivatives of arginine.
EP-A1-0,530,167 describing a:-alkoxy ketone derivatives of arginine as thrombin inhibitors.
In vivo diagnostic imaging methods for intravascular thrombi have been previously reported. These imaging methods use compounds that are detectably labeled with radioactive or paramagnetic atoms. For example, platelets labeled with the gamma emitter, In-111, can be employed as an imaging agent for detecting thrombi (Thakur, M. L. et al., Thromb Res. 9:345 (1976); Powers et al., Neurology 32:938 (1982)). The thrombolytic enzyme streptokinase labeled with Tc-99m has been proposed as an imaging agent (Wong, U.S. Pat. No. 4,418,052 (1983)). The fibrin-binding domains of Staphylococcus aureus derived protein A labeled with the gamma emitters, I-125 and I-131, have been proposed as imaging agents (Pang, U.S. Pat. No. 5,011,686 (1991)). Radiolabeled and paramagnetically labeled alpha-ketoamide derivatives have also been proposed as thrombus imaging agents (Abelman et al., U.S. Pat. No. 5,656,600).
A need continues to exist for non-peptidic compounds that are potent and selective protease inhibitors, and which possess greater bioavailability and fewer side-effects than currently available protease inhibitors. Accordingly, new classes of potent protease inhibitors, characterized by potent inhibitory capacity and low mammalian toxicity, are potentially valuable therapeutic agents for a variety of conditions, including treatment of a number of mammalian proteolytic disease states.
The present invention is directed to novel compounds having Formula I (below). Also provided are processes for preparing compounds of Formula I. The novel compounds of the present invention are potent inhibitors of proteases, especially trypsin-like serine proteases, such as chymotrypsin, trypsin, thrombin, plasmin and factor Xa. Certain of the compounds exhibit antithrombotic activity via direct, selective inhibition of thrombin, or are intermediates useful for forming compounds having antithrombotic activity.
The invention includes a composition for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compositions may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents. The compositions can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions.
Also provided are methods of inhibiting or treating aberrant proteolysis in a mammal, and methods for treating myocardial infarction; unstable angina; stroke; restenosis; deep vein thrombosis; disseminated intravascular coagulation caused by trauma, sepsis or tumor metastasis; hemodialysis; cardiopulmonary bypass surgery; adult respiratory distress syndrome; endotoxic shock; rheumatoid arthritis; ulcerative colitis; induration; metastasis; hypercoagulability during chemotherapy; Alzheimer""s disease; Down""s syndrome; fibrin formation in the eye; and wound healing. Other uses of compounds of the invention are as anticoagulants either embedded in or physically linked to materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters, blood dialysis machines, blood collection syringes and tubes, blood lines and stents.
The invention also includes a method for reducing the thrombogenicity of a surface in a mammal by attaching to the surface, either covalently or noncovalently, a compound of the invention.
In another aspect, the present invention includes compositions which are useful for in vivo imaging of thrombi in a mammal, comprising a compound of the present invention which is capable of being detected outside the body. Preferred are compositions comprising a pharmaceutically acceptable carrier and a diagnostically effective amount of a compound of the present invention and a detectable label, such as a radioactive atom.
In another aspect, the present invention includes methods which are useful for in vivo imaging or thrombi in a mammal.
A first aspect of the present invention is directed to compounds of the general Formula I, including stereoisomers: 
or a solvate, hydrate or pharmaceutically acceptable salt thereof; wherein:
A1 represents a structural fragment of Formula IIa, IIb, IIc, IId, IIe, IIf or IIg: 
xe2x80x83wherein:
k is an integer 0, 1, 2, 3 or 4;
j is an integer 1, 2, 3 or 4;
q is an integer 0, 1, 2 or 3;
R1 represents H, C1-4 alkyl or R11OOCxe2x80x94(C1-4)alkyl-, optionally substituted in the position which is alpha to the carbonyl group, with a group R14xe2x80x94(CH2)pxe2x80x94, wherein p is 0, 1 or 2 and R14 is methyl, phenyl, OH, COOR12, CONHR12, where R12 is H or C1-4 alkyl group, and R11 is H, C1-6 alkyl, or benzyl substituted in the 4-position by COOR12, where R12 is as defined above, or
R1 represents R13xe2x80x94NHxe2x80x94COxe2x80x94(C1-4)alkyl-, optionally substituted alpha to the carbonyl with C1-4 alkyl and where R13 is H, C1-4 alkyl or xe2x80x94CH2COOR12, where R12 is as defined above, or
R1 represents R12OOCxe2x80x94CH2xe2x80x94OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is optionally substituted alpha to the carbonyl with C1-4 alkyl and where R12 is as defined above, or
R1 represents C1-4 alkylsulfonyl, Ph(4-COOR12)xe2x80x94SO2xe2x80x94, Ph(3-COOR12)xe2x80x94SO2xe2x80x94, Ph(2-COOR12)xe2x80x94SO2xe2x80x94, where R12 is as defined above, or
R1 represents C1-4 alkylcarbonyl, or
R1 represents C1-4 alkoxycarbonyl, or
R1 represents xe2x80x94COxe2x80x94(CH2)pxe2x80x94COOR12, where R12 is as defined above and p is an integer 0, 1 or 2, or
R1 represents xe2x80x94CH2PO(OR15)2, xe2x80x94CH2SO3H or xe2x80x94CH2-(5-(1H)-tetrazolyl), where R15 is, individually at each occurrence, H, methyl or ethyl;
R2 represents H or C1-4 alkyl, carboxy(C1-4)alkyl or C1-4 alkoxycarbonyl(C1-4)alkyl;
R3 represents C1-4 alkyl, optionally having one or more fluorine atoms, or
R3 represents cyclopentyl, cyclohexyl or phenyl, any of which may be optionally substituted with C1-4 alkyl, or
R3 represents fluoren-9-yl, or 9-hydroxyfluoren-9-yl, or
R3 represents a phenyl group substituted with one to three OR16, where
R16 is independently H or C1-4 alkyl and k is 0, 1, or
R3 represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or
R3 represents a cis- or trans-decalin group and k is 0, 1, or
R3 represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl, any of which is optionally substituted with OR16, where R16 is as defined above and k is 0, 1, or
R3 represents Si(Me)3 or CH(R17)2, wherein R17 is independently C1-4 alkyl, cyclopentyl, cyclohexyl, benzyl or phenyl, or, in Formula IIa, where one R17 is cyclopentyl, cyclohexyl or phenyl, and the other R17 forms an ethylene bridge together with R1 and k is 0, 1, or 2;
R5 represents C1-4 alkyl, phenyl, or benzyl;
R21 represents H, C(O)R41, SiR42R43R44 or C1-6 alkyl which latter group is optionally substituted or terminated by one or more substituents selected from OR45 or (CH2)tR46;
R42, R43 and R44 independently represent H, phenyl or C1-6 alkyl;
R46 represents C1-4 alkyl, phenyl, OH, C(O)OR47 or C(O)N(H)R48;
R48 represents H, C1-4 alkyl or CH2C(O)OR49;
R45 and R47 independently represent H, C1-4 alkyl or C7-9 alkylphenyl;
R41 and R49 independently represent H or C1-4 alkyl; and t represents 0, 1 or 2;
R22 and R23 independently represent H, C1-4 alkyl, cyclohexyl or phenyl;
R24 represents a structural fragment of Formula IVa, IVb or IVc, 
xe2x80x83wherein
v, w and u independently represent 0, 1, 2, 3 or 4;
R34 and R35 independently represent H, Si(Me)3, 1- or 2-naphthyl, a polycyclic hydrocarbyl group, CHR31R32 or C1-4 alkyl (which latter group is optionally substituted by one or more fluorine atoms), or C3-8 cycloalkyl, phenyl, methylenedioxyphenyl, benzodioxanyl, benzofranyl, dihydrobenzofuranyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, coumaranonyl, coumarinyl or dihydrocoumarinyl (which latter twelve groups are optionally substituted by one or more of C1-4 alkyl (which latter group is optionally substituted by one or more halo substituent), C1-4 alkoxy, halo, hydroxy, cyano, nitro, SO2NH2, C(O)OH or N(H)R33); R31 and R32 independently represent cyclohexyl or phenyl; R36 and R37 independently represent H, C1-4 alkyl, C3-8 cycloalkyl, phenyl (which latter group is optionally substituted by one or more of C1-4 alkyl (which latter group is optionally substituted by one or more halo substituent), C1-4 alkoxy, halo, hydroxy, cyano, nitro, SO2NH2, C(O)OH or N(H)R38) or together with the carbon atom to which they are attached form a C3-8 cycloalkyl ring; R33 and R38 independently represent H or C(O)R39; and R39 represents H, C1-4 alkyl or C1-4 alkoxy;
A2 represents a structural fragment of Formula IIIa, IIIb or IIIc: 
xe2x80x83wherein
d is 0, 1 or 2;
e is 1, 2, 3 or 4;
Y represents a methylene group, or
Y represents an ethylene group and the resulting 5-membered ring may optionally carry one or two fluorine atoms, a hydroxy group or an oxo group in position 4, or may or may not be unsaturated, or
Y represents xe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94SOxe2x80x94, with the heteroatom functionality in position 4, or
Y represents a n-propylene group and the resulting 6-membered ring may optionally carry in position 5 one fluorine atom, a hydroxy group or an oxo group, carry two fluorine atoms in one of positions 4 or 5 or be unsaturated in position 4 and 5, or carry in position 4 a C1-4 alkyl group, or
Y represents xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94SOxe2x80x94CH2xe2x80x94, or
Y represents xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94;
R4 is as defined as for R3 above;
R6 represents H or C1-4 alkyl, carboxy, C1-4 alkoxycarbonyl, carboxy(C1-4)alkyl or C1-4 alkoxycarbonyl(C1-4)alkyl;
provided that when A1 is a fragment of Formula IIb, and A2 is a fragment of Formula IIIb, then R4 is not 1-naphthyl or 2-naphthyl;
R7 is one of hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, diakylariinoalkyl, carboxyalkyl, hydroxy, alkoxy, aralkoxy, aryloxy, heteroaryloxy, or mono- or di-alkylamino, provided that n is other than zero when R7 is hydroxy, alkoxy, aralkoxy, aryloxy, heteroaryloxy, or mono- or di-alkylamino;
R8, R9 and R10 are each independently one of hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or carboxyalkyl;
or R7 and R8 are taken together to form xe2x80x94(CH2)ixe2x80x94, where i is zero (a bond), 1 or 2, while R9 and R10 are defined as above; or R7 and R10 are taken together to form xe2x80x94(CH2)jxe2x80x94, where j is zero (a bond), or 1 to 8, while R8 and R9 are defined as above; or R9 and R10 are taken together to form xe2x80x94(CH2)hxe2x80x94, where h is 2-8, while R7 and R8 are defined as above;
R18 is one of hydrogen, alkyl, alkenyl, alkynyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylamino(C2-10)alkyl, dialkylamino(C2-10)alkyl or carboxyalkyl, or alternatively, R18 and R10 taken together to form xe2x80x94(CH2)wxe2x80x94, where w is 1-5;
X is oxygen, NR19; or CHxe2x95x90NR19, where the nitrogen of CHxe2x95x90NR19 is attached to the nitrogen of NR18;
R19 is one of hydrogen, alkyl, cycloalkyl or aryl, wherein said alkyl, cycloalkyl or aryl can be optionally substituted with amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, aryl, heteroaryl, acylamino, cyano or trifluoromethyl;
Ra, Rb and Rc are independently hydrogen, alkyl, hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano or xe2x80x94CO2Rw;
Rw is alkyl, cycloalkyl, phenyl, benzyl, 
xe2x80x83where Rd and Re are independently hydrogen, C1-6 alkyl, C2-6 alkenyl or phenyl, Rf is hydrogen, C1-6 alkyl, C2-6 alkenyl or phenyl, Rg is hydrogen, C1-6 alkyl, C2-6 alkenyl or phenyl, and Rh is aralkyl or C1-6 alkyl;
n is from zero to 8; and
m is from zero to 4.
Compounds of Formula I having S-configuration on the A2 fragment are preferred ones. Compounds also having R-configuration on the A1 fragment are particularly preferred ones.
The wavy lines on the carbon atom in the carbonyl group in Formulae IIa, IIb, IIc, IId, IIe, IIf, IIg, IIIa, IIIb, IIIc, and on the nitrogen atom in Formulae IIIa, IIIb, IIIc signify the bond position of the fragment.
The dots adjacent to the carbon atoms in fragments of Formula IVa, IVb and IVc signify the point of attachment of the fragments to the compound of Formula I.
Abbreviations are listed at the end of this specification.
Preferred values of R7, R8, R9 and R10 are independently one of hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, C6-10 aryl, C2-10 hydroxyalkyl or C2-7 carboxyalkyl. Useful values of R7, R8, R9 and R10 include hydrogen, methyl, ethyl, propyl, n-butyl, benzyl, phenylethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-carboxymethyl, 3-carboxyethyl and 4-carboxypropyl. Additional preferred compounds are those wherein R7 and R8 are taken together to form (xe2x80x94CH2)ixe2x80x94 where i is preferably 2, 3 or 4. Another group of preferred compounds are those where R8 and R9 are taken together to form xe2x80x94(CH2)hxe2x80x94 where h is most preferably 2.
R18 preferably represents H or C1-4 alkyl, carboxy, C1-4 alkoxycarbonyl, carboxy(C1-4)alkyl, C1-4 alkoxycarbonyl(C1-4)alkyl, (C6-10)ar(C1-6)alkyl, or C3-6alkenyl. Most preferred values of R18 are hydrogen or C1-6 alkyl or alternatively, R18 and R10 taken together to form xe2x80x94(CH2)wxe2x80x94, where w is 1-5.
A preferred value of X is O.
Also preferred is when X is CHxe2x95x90NR19, where R19 is preferably hydrogen, C1-6 alkyl, optionally substituted with amino, mono(C1-4)alkylamino, C1-6 alkoxy, hydroxy, carboxy, phenyl, C1-4 alkyloxycarbonyl, C6-10 ar(C1-4)alkoxycarbonyl, C1-6acylamino, cyano, or trifluoromethyl.
Preferred values of Ra, Rb and Rc in Formula I are hydrogen, hydroxy, C1-6alkyl, C1-6 alkoxy, cyano or xe2x80x94CO2Rw, where Rw, in each instance, is preferably one of C1-4 alkyl, C4-7 cycloalkyl or benzyloxycarbonyl. Suitable values of Ra, Rb and Rc include hydrogen, methyl, ethyl, propyl, n-butyl, hydroxy, methoxy, ethoxy, cyano, xe2x80x94CO2CH3, xe2x80x94CO2CH2CH3 and xe2x80x94CO2CH2CH2CH3. In the most preferred embodiments, Ra, Rb and Rc are each hydrogen.
Also preferred at Ra, Rb and Rc is the group xe2x80x94CO2Rw, where Rw is one of 
where Rd-Rh are defined as above. When Ra, Rb and Rc are xe2x80x94CO2Rw, where Rw is one of one of these moieties, the resulting compounds are prodrugs that possess desirable formulation and bioavailability characteristics. A preferred value for each of Rd, Re and Rg is hydrogen, Rf is methyl, and preferred values for Rh include benzyl and tert-butyl.
Preferred values of n in Formula I include from zero to 6, more preferably from zero to 4, and most preferably zero, 1 or 2. Preferred values of m include from zero to 4, more preferably zero, 1, 2 or 3.
According to the invention it has been found that compounds of the general Formula I, either as such or in the form of physiologically acceptable salts, and including stereoisomers, are potent serine protease inhibitors, wherein:
A1 represents a structural fragment of Formula IIa, IIb, IIc, IId or IIg, preferably IIa, IIb or IIg; wherein:
k is an integer 0, 1, 2, 3 or 4, preferably 0, 1;
q is an integer 0, 1, 2 or 3, preferably 1;
R1 represents H, C1-4 alkyl, R11OOCxe2x80x94(C1-4)alkyl-, optionally substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R14xe2x80x94(CH2)pxe2x80x94, wherein p is 0, 1 or 2 and R14 is methyl, phenyl, OH, COOR12, CONHR12, where R12 is H or C1-4 alkyl, and R11 is H or C1-6 alkyl, or
R1 represents Ph(4-COOR12)xe2x80x94CH2xe2x80x94, where R12 is as defined above, or
R1 represents R13xe2x80x94NHxe2x80x94COxe2x80x94(C1-4)alkyl-, and is optionally substituted alpha to the carbonyl with C1-4 alkyl, and where R13 is H or C1-4 alkyl or xe2x80x94CH2COOR12 where R12 is as defined above, or
R1 represents R12OOCxe2x80x94CH2xe2x80x94OOCxe2x80x94(C1-4)alkyl-, where the alkyl is optionally substituted alpha to the carbonyl with C1-4 alkyl and where R12 is as defined above, or
R1 represents C1-4 alkylsulfonyl, Ph(4-COOR12)xe2x80x94SO2xe2x80x94, Ph(3-COOR12)xe2x80x94SOxe2x80x94, Ph(2-COOR12)xe2x80x94SO2xe2x80x94 where R12 is as defined above or
R1 represents C1-4 alkylcarbonyl, or
R1 represents C1-4 alkoxycarbonyl, or
R1 represents xe2x80x94COxe2x80x94(CH2)pxe2x80x94COOR12, where R12 is as defined above and p is an integer 0, 1 or 2, or
R1 represents xe2x80x94CH2PO(OR15)2, xe2x80x94CH2SO3H or xe2x80x94CH2xe2x80x94(5-(1H)-tetrazolyl), where R15 is, individually at each occurrence, H, methyl or ethyl;
Preferably R1 represents R11OOCxe2x80x94(C1-4)alkyl-, and R11 is H.
R2 represents H or C1-4 alkyl, carboxy(C1-4)alkyl or C1-4alkoxycarbonyl(C1-4)alkyl;
R3 represents C1-4 alkyl, optionally substituted by one or more fluorine atoms, or
R3 represents cyclopentyl, cyclohexyl or phenyl, any of which may be optionally substituted with C1-4 alkyl, or
R3 represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or
R3 represents a cis- or trans-decalin group and k is 0, 1, or
R3 represents Si(Me)3 or CH(R17)2, wherein R17 is independently propyl, cyclopentyl, cyclohexyl, benzyl, or phenyl, or
R3 represents fluoren-9-yl or 9-hydroxy-fluoren-9-yl;
A2 represents a structural fragment of Formula IIIa, IIIb or IIIc, preferably IIIa; wherein:
d is an interger 0, 1 or 2;
e is an integer 1, 2, 3 or 4, preferably 2, or 3;
Y represents a methylene group, or
Y represents an ethylene group and the resulting 5-membered ring may optionally carry one or two fluorine atoms, a hydroxy group or an oxo group in position 4, or may optionally be unsaturated, or
Y represents xe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94SOxe2x80x94, with the heteroatom functionality in position 4, or
Y represents a n-propylene group and the resulting 6-membered ring may optionally carry in position 5 one fluorine atom, a hydroxy group or an oxo group, carry two fluorine atoms in one of positions 4 or 5 or be unsaturated in position 4 and 5, or carry in position 4 a C1-4 alkyl group, or
Y represents xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94SOCH2xe2x80x94, or
Y represents xe2x80x94CH2xe2x80x94CH2CH2xe2x80x94CH2xe2x80x94;
R4 represents C1-4 alkyl, or
R4 represents a Si(Me)3 group;
R6 represents H or C, alkyl, preferably H or a methyl group, or
R6 represents xe2x80x94(CH2)pxe2x80x94COOR51, where p is 0, 1 or 2 and R51 is H or C1-4alkyl, preferably p is 0 and R51 is H;
Ra, Rb and Rc are each one of hydrogen, C1-4 alkyl, hydroxy, C1-4 alkoxy, phenoxy, C1-4 alkyloxycarbonyl, benzyloxycarbonyl, cyano, 
xe2x80x83where Rh is benzyl, methyl, ethyl, isopropyl, sec-butyl or t-butyl, and where Rf is hydrogen or C1-6 alkyl;
R7, R8, R9 and R10 are independently one of hydrogen, C1-6 alkyl, C2-10carboxyalkyl or C2-10 hydroxyalkyl, or R7 and R8 are taken together to form xe2x80x94(CH2)ixe2x80x94 where i is zero, 1 or 2, while R9 and R10 are defined as above; or R7 and R10 are taken together to form xe2x80x94(CH2)jxe2x80x94, where j is zero (a bond), or 1, 2 or 3, while R8 and R9 are defined as above; or R9 and R10 are taken together to form xe2x80x94(CH2)hxe2x80x94, where h is 2, 3, or 4, while R7 and R8 are defined as above;
R18 is one of hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, C6-10 aryl, C2-10 hydroxyalkyl, C2-10 aminoalkyl, mono(C1-4)alkylamino(C2-8)alkyl, di(C1-4)alkylamino(C2-8)alkyl or C2-10 carboxyalkyl, or alternatively, R18 and R10 taken together to form xe2x80x94(CH2)wxe2x80x94, where w is 1-5;
X is xe2x80x94Oxe2x80x94, xe2x80x94NR19 or xe2x80x94CHxe2x95x90NR19xe2x80x94;
R19 is hydrogen or C1-4 alkyl;
n is from zero to 4; and m is from zero to 4.
According to a preferred embodiment the invention relates to compounds of Formula I, wherein:
A1 represents a structural fragment of Formula IIa, wherein:
k is 0 or 1;
R1 represents R11OOCxe2x80x94(C1-4)alkyl-, particularly methylene, ethylene and R11 is H;
R2 represents H;
R3 represents a cyclohexyl group;
A2 represents a structural fragment of Formula IIIa, wherein:
Y represents a methylene group, an ethylene group, or a n-propylene group and the resulting 6-membered ring may optionally carry in position 4 a C1-4alkyl group, preferably Y represents methylene, ethylene; and
R6 represents H;
Ra, Rb and Rc are hydrogen, hydroxy, 
xe2x80x83where Rh is benzyl or t-butyl, and where Rf is hydrogen or methyl;
R7, R8, R9 and R10 are independently one of hydrogen, C1-6 alkyl, C2-10hydroxyalkyl or C2-10 carboxyalkyl, or R7 and R8 are taken together to form (CH2)ixe2x80x94 where i is zero, 1 or 2, while R9 and R10 are defined as above; or R7 and R10 are taken together to form xe2x80x94(CH2)jxe2x80x94, where j is zero (a bond), or 1, 2 or 3, while R8 and R9 are defined as above; or R9 and R10 are taken together to form xe2x80x94(CH2)hxe2x80x94, where h is 2, 3 or 4, while R7 and R8 are defined as above;
R18 is hydrogen, C1-4 alkyl, C2-4 hydroxyalkyl, C2-4 carboxyalkyl, C2-4aminoalkyl, dimethylamino(C2-8)alkyl, or methylamino(C2-8)alkyl;
X is xe2x80x94Oxe2x80x94, xe2x80x94NR19xe2x80x94 or xe2x80x94CHxe2x95x90NR19xe2x80x94, preferably O;
R19 is hydrogen, or C1-6 alkyl;
n is from zero to 4; and
m is zero, 1, 2 or 3.
Additional preferred compounds of Formula I include those wherein:
A1 represents a structural fragment of Formula IIf;
R21 represents optionally substituted C1-6 alkyl or, particularly, H;
R24 represents a structural fragment of Formula IVa;
A2 represents a structural fragment of Formula IIa;
Y represents CH2 or (CH2)2xe2x80x94; and
n represents 1.
Additional preferred compounds of Formula I include those wherein:
A1 represents a structural fragment of Formula IIg;
k is zero or 1;
R3 represents phenyl or benzyl, optionally substituted by one to three of OR16, where R16 is hydrogen or methyl, or
R3 represents fluoren-9-yl or 9-hydroxyfluoren-9-yl, or
R3 represents CH(R17)2, where R17 is cyclohexyl or phenyl;
A2 represents a structural fragment of Formula IIIa;
Y represents CH2 or (CH2)2;
n represents 1.
Compounds of Formula I having A2 as a fragment 
in the S-configuration are preferred. The wavy lines on the nitrogen and carbon atom in the above fragment signify the bond position of the fragment.
Most preferred compounds of the present invention have one of the Formulae Ia, Ib or Ic: 
or a pharmaceutically acceptable salt thereof; where:
nxe2x80x2 is 1, 2 or 3, preferably 1 or 2;
nxe2x80x3 is 0, 1, 2 or 3, preferably 0 or 1;
mxe2x80x2 is 0, 1, 2 or 3, preferably 0 or 1;
Xxe2x80x2 is xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94 or xe2x80x94CHxe2x95x90NHxe2x80x94 (an amidinohydrazone group).
In this aspect of the present invention, combinations of A1 and A2 result in the following preferred A1xe2x80x94A2xe2x80x94 fragments:
HOOCxe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cgl-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cgl-Proxe2x80x94
(HOOCxe2x80x94CH2)2xe2x80x94(R)Cgl-Proxe2x80x94
Hxe2x80x94(R)Cgl-Picxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R,S)CH(COOH)xe2x80x94(R)Cgl-Picxe2x80x94
Hxe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(RorS)CH(COOH)xe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2CH2xe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94(R)Cha-Azexe2x80x94
Hxe2x80x94(R)Cha-Pro-Pabxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2-(Me)(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2-(Me)(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(RorS)CH(COOH)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94NHxe2x80x94COxe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
EtOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
Ph(4-COOH)xe2x80x94SO2xe2x80x94(R)Cha-Proxe2x80x94
Hxe2x80x94(R)Cha-Picxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Picxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(RorS)CH(COOH)xe2x80x94(R)Cha-Picxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Picxe2x80x94
HOOCxe2x80x94COxe2x80x94(R)Cha-Picxe2x80x94
HOOCxe2x80x94CHxe2x80x94COxe2x80x94(R)Cha-Picxe2x80x94
Me-OOCxe2x80x94CH2xe2x80x94COxe2x80x94(R)Cha-Picxe2x80x94
H2Nxe2x80x94COxe2x80x94CH2xe2x80x94(R)Cha-Picxe2x80x94
Boc-(R)Cha-Picxe2x80x94
Ac-(R)Cha-Picxe2x80x94
Me-SO2xe2x80x94(R)Cha-Picxe2x80x94
Hxe2x80x94(R)Cha(R,S)betaPicxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-(R,S)betaPicxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Valxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Valxe2x80x94
Hxe2x80x94(R)Hoc-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Hoc-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R,S)CH(COOH)xe2x80x94(R)Hoc-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Hoc-Picxe2x80x94
(HOOCxe2x80x94CH2)2xe2x80x94(R)Hoc-Picxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Pro(3-(S)Ph)-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Pro(3-S)Ph)-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Tic-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cgl-Proxe2x80x94
Hxe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
Hxe2x80x94(R)Cha-Proxe2x80x94
Hxe2x80x94(R)Cgl-Ilexe2x80x94
Hxe2x80x94(R)Cgl-Azexe2x80x94
HOOCxe2x80x94(R,S)CH(Me)-(R)Cha-Proxe2x80x94
MeOOCxe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
EtOOCxe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
nBuOOCxe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
nHexOOCxe2x80x94CH2xe2x80x94(R)Cgl-Azexe2x80x94
Hxe2x80x94(R)Cgl-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cgl-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Azexe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
(HOOCxe2x80x94CH2)2xe2x80x94(R)Cgl-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2(HOOCxe2x80x94CH2)xe2x80x94(R)Cha-Proxe2x80x94
Hxe2x80x94(R)Phe-Chaxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Phe-Chaxe2x80x94
Hxe2x80x94(R)Cha-Chaxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Chaxe2x80x94
Hxe2x80x94(R)Cha-Proxe2x80x94
Me-(R)Cha-Proxe2x80x94
HOxe2x80x94(CH2)3xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
iPrOOCxe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2-(Me)(R)Cha-Proxe2x80x94
HOOCxe2x80x94(R,S)CH(Me)-(R)Cha-Proxe2x80x94
HOOCxe2x80x94(R,S)CH(CH2CH2Ph)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
EtOOCxe2x80x94COxe2x80x94(R)Cha-Proxe2x80x94
(R,S)Bla-(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(nBu)(R)Cha-Proxe2x80x94
HOOCxe2x80x94(R,S)CH(Me)-(R)Cha-Proxe2x80x94
EtOOCxe2x80x94(R,S)CH(Me)HR)Cha-Proxe2x80x94
HOOCxe2x80x94(R)CH(CH2xe2x80x94OH)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94(R,S)CH(Ph)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94(S)CH(CH2CH2Ph)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94(R)CH(CH2CH2Ph)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94COxe2x80x94(R)Cha-Proxe2x80x94
MeOOCxe2x80x94COxe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94(R,S)CH(CH2COOH)xe2x80x94(R)Cha-Proxe2x80x94
MeOOCxe2x80x94(R,S)CH(CH2COOMe)xe2x80x94(R)Cha-Proxe2x80x94
HOOCxe2x80x94Ph-4-CH2xe2x80x94R)Cha-Proxe2x80x94
(HO)2P(O)xe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
EtO(HO)P(O)H2xe2x80x94(R)Cha-Proxe2x80x94
(EtO)2P(O)xe2x80x94CH2xe2x80x94(R)Cha-Proxe2x80x94
Hxe2x80x94(R,S)Pro(3-(trans)Ch)-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R,S)Pro(3-(trans)Ph)-Proxe2x80x94
N-fluoren-9-ylcarboxy-Proxe2x80x94
N-(9-hydroxyfluoren-9-ylcarboxy)-Proxe2x80x94
Dca-Proxe2x80x94
Boc-Dca-Proxe2x80x94
Dpa-Proxe2x80x94
Boc-Dpa-Proxe2x80x94
(Ph)2CHCH2C(O)-Proxe2x80x94
(Ph)2CHC(O)-Proxe2x80x94
(Chx)2CHCH2C(O)-Proxe2x80x94
(Chx)2CHC(O)Proxe2x80x94
Of those fragments, the following fragments are most preferred:
HOOCCH2-(Me)(R)Cha-Proxe2x80x94
HOOCxe2x80x94CH2xe2x80x94(R)Cha-Picxe2x80x94
Alternative embodiments of the present invention include compounds of Formula I in which two xe2x80x9cRxe2x80x9d groups together form a saturated or unsaturated hydrocarbon bridge, thus forming an additional cyclic moiety in the resulting compounds. Alternative embodiments include compounds of Formula I wherein A1, A2, m and n are as defined above; and,
A. R18 and Rb are taken together to form xe2x80x94(CH2)xe2x80x94(CH2)r or xe2x95x90CHxe2x80x94Nxe2x95x90CHxe2x80x94NHxe2x80x94, where r is 1, 2 or 3;
Ra is hydrogen or hydroxy;
Rc is hydrogen, alkyl, hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbamoyloxy, cyano or xe2x80x94CO2Rwxe2x80x94, where Rw is as defined above;
R7, R8, R9 and R10 are independently one of hydrogen, C1-6 alkyl, C2-10 carboxyalkyl or C2-10 hydroxyalkyl, or R7 and R8 are taken together to form xe2x80x94(CH2)ixe2x80x94 where i is zero, 1 or 2, while R9 and R10 are defined as above; or R7 and R10 are taken together to form xe2x80x94(CH2)jxe2x80x94, where j is zero (a bond), or 1, 2 or 3, while R8 and R9 are defined as above; or R9 and R10 are taken together to form xe2x80x94(CH2)hxe2x80x94, where h is 2, 3, or 4, while R7 and R8 are defined as above; or
B. Ra and Rc are taken together to form xe2x80x94CH2xe2x80x94(CH2)sxe2x80x94, where s is 1 or 2;
R18 is hydrogen, alkyl, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano or xe2x80x94CO2Rwxe2x80x94, where Rw is as defined above; and
R7, R8, R9 and R10 are independently one of hydrogen, C1-6 alkyl, C2-10 carboxyalkyl or C2-10 hydroxyalkyl, or R7 and R8 are taken together to form xe2x80x94(CH2)ixe2x80x94 where i is zero, 1 or 2, while R9 and R10 are defined as above; or R7 and R10 are taken together to form xe2x80x94(CH2)jxe2x80x94 where j is zero (a bond), or 1, 2 or 3, while R8 and R9 are defined as above; or R9 and R10 are taken together to form xe2x80x94(CH2)hxe2x80x94, where h is 2, 3, or 4, while R7 and R8 are defined as above.
It is also to be understood that the present invention is considered to include stereoisomers as well as optical isomers, e.g. mixtures of enantiomers as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in selected compounds of the present series.
The compounds of Formula I may also be solvated, especially hydrated. Hydration may occur during manufacturing of the compounds or compositions comprising the compounds, or the hydration may occur over time due to the hygroscopic nature of the compounds.
Certain compounds within the scope of Formula I are derivatives referred to as prodrugs. The expression xe2x80x9cprodrugxe2x80x9d denotes a derivative of a known direct acting drug, which derivative has enhanced delivery characteristics and therapeutic value as compared to the drug, and is transformed into the active drug by an enzymatic or chemical process; see Notari, R. E., xe2x80x9cTheory and Practice of Prodrug Kinetics,xe2x80x9d Methods in Enzymology, 112:309-323 (1985); Bodor, N., xe2x80x9cNovel Approaches in Prodrug Design,xe2x80x9d Drugs of the Future, 6(3):165-182 (1981); and Bundgaard, H., xe2x80x9cDesign of Prodrugs: Bioreversible-Derivatives for Various Functional Groups and Chemical Entities,xe2x80x9d in Design of Prodrugs (H. Bundgaard, ed.), Elsevier, New York (1985). Useful prodrugs are those where Ra, Rb and/or Rc are xe2x80x94CO2Rw, where Rw is defined above. See, U.S. Pat. No. 5,466,811 and Saulnier et al., Bioorg. Med. Chem. Lett. 4:1985-1990 (1994).
In another aspect, the present invention includes compositions which are useful for in vivo imaging of thrombi in a mammal, comprising a compound of the present invention which is capable of being detected outside the body. Preferred are compositions comprising a compound of the present invention and a detectable label, such as a radioactive atom.
In another aspect, the present invention provides diagnostic compositions which are used for in vivo imaging of thrombi in a mammal, comprising a pharmaceutically acceptable carrier and a diagnostically effective amount of a compound or composition of the present invention.
In another aspect, the present invention includes methods which are useful for in vivo imaging or thrombi in a mammal.
According to a preferred aspect, useful compounds are those wherein the R1 or R3 substituent is substituted with a detectable label, such as a radioactive iodine atom, such as I-125, I-131 or I-123. In this aspect, R1 is preferably phenyl, having a para I-123, para I-125 or para I-131 substitution, or benzyl, having a meta I-123, meta I-125 or meta I-131 substitution.
The term xe2x80x9calkylxe2x80x9d as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 12 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl.
The term xe2x80x9calkenylxe2x80x9d is used herein to mean a straight or branched chain radical of 2-20 carbon atoms, unless the chain length is limited thereto, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Preferably, the alkenyl chain is 2 to 10 carbon atoms in length, more preferably, 2 to 8 carbon atoms in length most preferably from 2 to 4 carbon atoms in length.
The term xe2x80x9calkynylxe2x80x9d is used herein to mean a straight or branched chain radical of 2-20 carbon atoms, unless the chain length is limited thereto, wherein there is at least one triple bond between two of the carbon atoms in the chain, including, but not limited to, acetylene, 1-propylene, 2-propylene, and the like. Preferably, the alkynyl chain is 2 to 10 carbon atoms in length, more preferably, 2 to 8 carbon atoms in length, most preferably from 2 to 4 carbon atoms in length.
In all instances herein where there is an alkenyl or alkynyl moiety as a substituent group, the unsaturated linkage, i.e., the vinylene or acetylene linkage is preferably not directly attached to a nitrogen, oxygen or sulfur moiety.
The term xe2x80x9calkoxyxe2x80x9d is used herein to mean a straight or branched chain radical of 1 to 20 carbon atoms, unless the chain length is limited thereto, bonded to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably the alkoxy chain is 1 to 10 carbon atoms in length, more preferably 1 to 8 carbon atoms in length.
The term xe2x80x9carylxe2x80x9d as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl or tetrahydronaphthyl.
The term xe2x80x9cheteroarylxe2x80x9d as employed herein refers to groups having 5 to 14 ring atoms; 6, 10 or 14xcfx80 electrons shared in a cyclic array; and containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms (where examples of heteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathienyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl, 4xcex1H-carbazolyl, carbazolyl, xcex2-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups).
The term xe2x80x9caralkylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d as employed herein by itself or as part of another group refers to C1-6 alkyl groups as discussed above having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.
The term xe2x80x9ccycloalkylxe2x80x9d as employed herein by itself or as part of another group refers to cycloalkyl groups containing 3 to 9 carbon atoms. Typical examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl.
The terms xe2x80x9calkoxyxe2x80x9d refers to any of the above alkyl groups linked to an oxygen atom.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine with chlorine being preferred.
The term xe2x80x9cmonoalkylarninexe2x80x9d as employed herein by itself or as part of another group refers to an amino group which is substituted with one alkyl group having from 1 to 6 carbon atoms.
The term xe2x80x9cdialkylaminexe2x80x9d as employed herein by itself or as part of another group refers to an amino group which is substituted with two alkyl groups, each having from 1 to 6 carbon atoms
The term xe2x80x9chydroxyalkylxe2x80x9d as employed herein refers to any of the above alkyl groups substituted by one or more hydroxyl moieties.
The term xe2x80x9ccarboxyalkylxe2x80x9d as employed herein refers to any of the above alkyl groups substituted by one or more carboxylic acid moieties.
The term xe2x80x9cheterocyclicxe2x80x9d is used herein to mean a saturated or wholly or partially unsaturated 3-7 membered monocyclic, or 7-10 membered bicyclic ring system, which consists of carbon atoms and from one to four heteroatoms independently selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, the nitrogen can be optionally quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring, and wherein the heterocyclic ring can be substituted on carbon or on a nitrogen atom if the resulting compound is stable. Especially useful are rings containing one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms. Examples of such heterocyclic groups include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyirolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydroturyl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same as morpholinyl.
The term xe2x80x9cheteroatomxe2x80x9d is used herein to mean an oxygen atom (xe2x80x9cOxe2x80x9d), a sulfur atom (xe2x80x9cSxe2x80x9d) or a nitrogen atom (xe2x80x9cNxe2x80x9d). It will be recognized that when the heteroatom is nitrogen, it may form an NRyRz moiety, wherein Ry and Rz are, independently from one another, hydrogen or C1 to C8 alkyl, or together with the nitrogen to which they are bound, form a saturated or unsaturated 5-, 6-, or 7-membered ring.
The term xe2x80x9cheteroarylxe2x80x9d includes 5 or 6 membered aromatic heterocyclic rings containing one or more heteroatoms selected from nitrogen, sulphur and oxygen atoms, and fused bicyclic ring systems containing one or more nitrogen, sulfur, and oxygen atoms. Examples of such groups include oxadiazole, thiazole thiadiazole, triazole, tetrazole, benzimidazole, pyridine, furan and thiophene.
A C3-7 cycloalkenyl group includes rings containing at least one double bond incorporated in the ring.
A C3-7 heterocycloalkyl group includes rings containing one or more heteroatoms selected from nitrogen, sulphur and oxygen atoms, for example, a tetrahydropyran-4-yl group.
A C3-7 heterocycloalkenyl group includes rings containing one or more heteroatoms selected from nitrogen, sulphur and oxygen atoms, together with at least on double bond incorporated in the ring.
Methods of Making
Preparation of Starting Materials/Protection Procedures/Deprotection Procedures
The following starting materials can be prepared by the methods described in U.S. Pat. No. 5,614,499, fully incorporated by reference herein:
Boc-(R)Pgl-OH
Boc-(R)Cha-OH
Boc-(R)Hop-OH
H-Pab(Z)xc3x97HCl
H-Pic-OEtxc3x97HCl
Boc-(R)Cgl-OH
Boc-(R)Hoc-OH
Boc-(R)Cgl-Aze-OH
Boc-(R)Cgl-Pic-OH
Boc-(R)Cgl-Pro-OH
Boc-(R)Cha-Aze-OH
Boc-(R)Cha-Pro-OH
Boc-(R)Cha-Pic-OH
Boc-(R)Cha-(R,S)betaPic-OH
Boc-(R)Cha-Val-OH
Boc-(R)Hoc-Aze-OH
Boc-(R)Hoc-Pro-OH
Boc-(R)Hoc-Pic-OH
Boc-(R)Pro-Phe-OH
Boc-(R)Pro(3-(S)Ph)-Pro-OH
Boc-(R)Tic-Pro-OH
Boc-(R)Cgl-Ile-OH
Boc-(R)Phe-Phe-OH
H-(R)Dph-OH
Boc-(R)Cgl-OH
Boc(R)Dch-OH
Boc-(Me)(R)Cha-OH
Boc-(R)Cha-Pro-OH
Boc-(R)Cha-Pic-OH
Boc-(R,S)Pro(3-(trans)Ph-Pro-OH
Boc-(R,S)Pro(3-(trans)Ch)-Pro-OH
Boc-(R)Hoc-OH
Boc-(R)Hoc-Pro-OH
Boc-(R)Hoc-Pic-OH
Boc-(R)Cha-Aze-OH
Boc-(R)Cha-Pic(4-(S)Me)-OH
Boc-(R)Cha-(R)Pic(4-(R)Me)-OH
Boc-(R)Cha-(R,S)Pic(4,5-dehydro)-OH
Boc-(R)Cgl-Pic-OH
Boc-(R)Dph-Pic-OH
Boc-(R)Dch-Pic-OH
Boc-(R)Cha-Pro(5-(S)Me)-OH
Procedures for protection and deprotection of functional groups in starting materials and intermediates are described in U.S. Pat. No. 5,614,499, fully incorporated by reference herein.
Synthesis
Another aspect of the present invention is a process for preparing an amidinohydrazone compound of Formula I, comprising reacting an ,aminoguanidine of the formula: 
wherein R18, Ra, Rb and Rc are defined as above, with a derivatized dipeptide of the formula: 
wherein Pb is an amino-protecting group, and A1, A2, R7, R8, R9, and n are defined as above to form an amidinohydrazone, of Formula IX: 
where Pb, A1, A2, R7xe2x80x94R9, R18, Ra, Rb, Rc and n are as defined above.
The aminoguanidine (Formula VII) is typically provided as a salt, preferably the nitrate salt. This step proceeds at ambient temperature using alcohol as a solvent. An acid, such as 4N HCl in dioxane is added to the reaction mixture. The reaction is more fully described herein.
The present invention is also directed to a process for preparing an aminoguanidine compound of Formula I, comprising selectively reducing the hydrazone carbon to nitrogen double bond of an amidinohydrazone of Formula IX. Pb is an N-terminal amino protecting group, such as tert-butyloxy carbonyl or benzyloxy-carbonyl.
Another aspect of the present invention is a process for preparing a alkoxyguanidine compound of Formula I, comprising reacting an alkoxyamine derivatized dipeptide of the formula: 
wherein Pb, A1, A2, R7xe2x80x94R10, n and m are defined as above with a guanidinylating reagent. Preferred guanidinylating reagents include: aminoiminosulfonic acid, optionally substituted 1H-pyrazole-1-carboxamidines, or N,Nxe2x80x2-bis(tert-butoxycarbonyl)S-methyl isothiourea.
In more detail, compounds of Formula I can be formed by the methods described in Schemes 4-6. Scheme 4 depicts a synthesis scheme for forming alkoxyguanidines (X is O) of Formula I, Scheme 5 depicts an alternate synthesis scheme for forming alkoxyguanidines of Formula I, and Scheme 6 depicts a synthesis scheme for forming aminoguanidines (X is NR19) or amidinohydrazones (X is CHxe2x95x90NR19) of Formula I With respect to these schemes, an N-terminally protected peptide, Paxe2x80x94A2 is coupled with an aminoalcohol using standard peptide coupling. The resulting intermediate is thereafter coupled to Pbxe2x80x94A1 using standard peptide coupling techniques. Alternatively, A1 and A2 can be coupled, prior to coupling of A2 and the aminoalcohol.
The alcohol is converted employing a Mitsunobu reaction with an N-hydroxycyclic imide derivative such as N-hydroxyphthalimide. Unveiling of the phthalimide protecting group is accomplished using standard conditions well known in the art (Greene, T. W. and Wuts, P. G. M., supra), for example, sodium borohydride in a mixture of an appropriate alcohol (e.g. ethanol or 2-propanol)/water followed by acidification. Alternatively, removal of the protecting group may be accomplished using hydrazine or methylamine.
Guanidinylation of the resulting alkoxy amine is achieved using standard reagents such as aminoiminosulfonic acid (Miller, A. E. and Bischoff, J. J. Synthesis 777 (1986)), or 1H-pyrazole-1-carboxamidines hydrochloride (Bernatowicz, M. S. et. al. J. Org. Chem. 57(8):2497 (1992)), or with substituted guanidinylating reagents such as N,Nxe2x80x2-bis(tert-butoxycarbonyl)-S-methylisothiourea (Bergeron, R. J. and McManis, J. S. J. Org. Chem. 52:1700 (1987)) or Nxe2x80x94Ra, Nxe2x80x94Rb, Nxe2x80x2Rc-1H-pyrazole-1-carboxamidine, where Ra, Rb and Rc are defined as above for Formula I. Useful 1H-pyrazole-1-carboxamidines include N,Nxe2x80x2-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine and N,Nxe2x80x2-bis(benzyloxycarbonyl)-1H-pyrazole-1-carboxamidine (all of which can be prepared according to Bernatowicz, M. S. et. al., Tetrahedron Letters 34:3389 (1993)).
Alternatively, peptide Paxe2x80x94A2 may be coupled directly to an aminoaldehyde or aminodiol using standard peptide coupling techniques. Or, dipeptide Pbxe2x80x94A1xe2x80x94A2 can be coupled to the aminoaldehyde or aminodiol.
The compound having a free aldehyde is then converted to amidinohydrazone using standard conditions, for example, treatment with an aminoguanidine, such as aminoguanidine or 2-hydrazinoimidazoline, optionally in the presence of an acid such as nitric acid, hydrogen chloride, or hydrogen bromide, in an appropriate solvent, for example, ethanol or methanol, which, in addition, may contain other solvents such as dichloromethane or tetrahydrofuran. Conversion of amidinohydrazone to aminoguanidine is accomplished under reducing conditions well known in the art, for example, lithium borohydride in an appropriate solvent such as tetrahydrofuran or methanol at various temperatures up to reflux. As an alternative method, catalytic hydrogenation with palladium on carbon catalyst can be employed.
When Ra, Rb and/or Rc are a protecting group, for example t-butyloxycarbonyl (Boc), these protecting groups can be optionally removed by treatment with acid, usually trifluoroacetic acid in a suitable solvent such as dichloromethane or water, or by HCl gas dissolved in a suitable solvent, such as 1,4-dioxane.
Compounds wherein Ra and Rc together form a cyclic group, such as an imidazoline, can be synthesized by employing an imidazoline in place of the aminoguanidine in the above schemes.
Compounds wherein R7 and R10 or R8 and R10 together form a methylene linkage can be synthesized by substituting a cyclic ketone having a reactive group L that is attached directly or indirectly to the carbocyclic ring. Examples of suitable reagents include 2-hydroxycyclopentanone, 3-hydroxycyclopentanone, 2-hydroxycyclohexanone and 3-hydroxycyclohexanone.
Compounds IX wherein R18 and Rb, or Ra and Rc are taken together with the nitrogens to which they are attached to form a ring structure are prepared by substituting a heterocyclic amine XI or XII (below) for the aminoguanidine in the above schemes. 
According to the invention there is also provided a process for the preparation of compounds of Formula I which comprises:
(a) the coupling of a compound of Formula XV, 
xe2x80x83wherein R21, R22, R23 and R24 are as hereinbefore defined with a compound of Formula XVI, 
xe2x80x83wherein Y, R6xe2x80x94R10, n, m, X, Ra, Rb and Rc are as hereinbefore defined; or
(b) the coupling of a compound of Formula XVII, 
xe2x80x83wherein R21, R22, R23, R24 and Y are as hereinbefore defined with a compound of Formula XVIII, 
xe2x80x83wherein R7xe2x80x94R10, R18, n, m, X, Ra, Rb and Rc are as hereinbefore defined, for example in the presence of a coupling system (e.g. oxalyl chloride in DMF, EDC, DCC, HATU, or BOP), an appropriate base (e.g. pyridine, DMAP, TEA or DIEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF).
Compounds of Formula XV are commercially available, are well known in the literature, or are available using known techniques.
It will be appreciated by those skilled in the art that in the process described above the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups which it is desirable to protect include hydroxy, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters. Suitable protecting groups for amino, amidino and guanidino include t-butyloxycarbonyl or benzyloxy carbonyl. Amidino and guanidino nitrogens may be either mono- or diprotected.
The protection and deprotection of functional groups may take place before or after coupling.
In particular, the compounds of Formula I may be prepared by processes comprising the coupling of an N-acylated amino acid or a N-protected amino acid. When a N-protected amino acid is used the acyl group may be added after coupling and deprotection of the nitrogen atom may then be effected using standard methods thereafter.
Protecting groups may be removed in accordance with techniques which are well known to those skilled in the art and as described hereinafter.
The use of protecting groups is fully described in xe2x80x98Protective Groups in Organic Chemistryxe2x80x99, edited by J. W. F. McOmie, Plenum Press (1973), and xe2x80x98Protective Groups in Organic Synthesisxe2x80x99, 2nd edition, T. W. Greene and P. G. M. Wuts, Wiley-Interscience (1991).
It will also be appreciated by those skilled in the art that, although such protected derivatives of compounds of Formula I may not possess pharmacological activity as such, they may be administered parenterally or orally and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as xe2x80x9cprodrugs.xe2x80x9d All prodrugs of compounds of Formula I are included within the scope of the invention.
Compounds of Formula I, pharmaceutically-acceptable salts, tautomers and stereoisomers thereof, as well as prodrugs thereof, are hereinafter referred to together as xe2x80x9cthe compounds of the invention.xe2x80x9d
Uses
For medicinal use, the pharmaceutically acceptable acid addition salts, those salts in which the anion does not contribute significantly to toxicity or pharmacological activity of the organic cation, are preferred. The acid addition salts are obtained either by reaction of an organic base of Formula I with an organic or inorganic acid, preferably by contact in solution, or by any of the standard methods detailed in the literature available to any practitioner skilled in the art. Examples of useful organic acids are carboxylic acids such as maleic acid, acetic acid, trifluoroacetic acid, tartaric acid, propionic acid, fumaric acid, isethionic acid, succinic acid, cyclamic acid, pivalic acid and the like; useful inorganic acids are hydrohalide acids such as HCl, HBr, HI; sulfuric acid; phosphoric acid and the like. Preferred acids for forming acid addition salts include HCl, trifluoroacetic acid, and acetic acid.
The compounds of the present invention represent a novel class of potent inhibitors of metallo, acid, thiol and serine proteases. Examples of the serine proteases inhibited by compounds within the scope of the invention include leukocyte neutrophil elastase, a proteolytic enzyme implicated in the pathogenesis of emphysema; chymotrypsin and trypsin, digestive enzymes; pancreatic elastase, and cathepsin G, a chymotrypsin-like protease also associated with leukocytes; thrombin and factor Xa, proteolytic enzymes in the blood coagulation pathway. Inhibition of thermolysin, a metalloprotease, and pepsin, an acid protease, are also contemplated uses of compounds of the present invention. The compounds of the present invention are preferably employed to inhibit trypsin-like proteases.
An end use application of the compounds that inhibit chymotrypsin and trypsin is in the treatment of pancreatitis. For their end-use application, the potency and other biochemical parameters of the enzyme-inhibiting characteristics of the compounds of the present invention is readily ascertained by standard biochemical techniques well known in the art. Actual dose ranges for their specific end-use application will, of course, depend upon the nature and severity of the disease state of the patient or animal to be treated, as determined by the attending diagnostician. It is expected that a useful dose range will be about 0.01 to 10 mg per kg per day for an effective therapeutic effect.
Compounds of the present invention that are distinguished by their ability to inhibit either factor Xa or thrombin may be employed for a number of therapeutic purposes. As factor Xa or thrombin inhibitors, compounds of the present invention inhibit thrombin production. Therefore, these compounds are useful for the treatment or prophylaxis of states characterized by abnormal venous or arterial thrombosis involving either thrombin production or action. These states include, but are not limited to, deep vein thrombosis; disseminated intravascular coagulopathy which occurs during septic shock, viral infections and cancer; myocardial infarction; stroke; coronary artery bypass; fibrin formation in the eye; hip replacement; and thrombus formation resulting from either thrombolytic therapy or percutaneous transluminal coronary angioplasty (PCTA).
Other uses include the use of said thrombin inhibitors as anticoagulants either embedded in or physically linked to materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters, blood dialysis machines, blood collection syringes and tubes, blood lines and stents. The compounds of the present invention may also be used as an anticoagulant in extracorporeal blood circuits.
Metal stents have been shown to reduce restenosis, but are thrombogenic. A strategy for reducing the thrombogenicity of stents is to coat, embed, adsord or covalently attach a thrombin-inhibiting agent to the stent surface. The compounds of the present invention can be employed for this purpose. Compounds of the invention can be attached to, or embedded within soluble and/or biodegradeable polymers as and thereafter coated onto stent materials. Such polymers can include polyvinylpyrrolidone, polyhydroxy-propylmethacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels. See European Application 761 251, European Application 604,022, Canadian Patent 2,164,684 and PCT Published Applications WO 96/11668, WO 96/32143 and WO 96/38136.
By virtue of the effects of both factor Xa and thrombin on a host of cell types, such as smooth muscle cells, endothelial cells and neutrophils, the compounds of the present invention find additional use in the treatment or prophylaxis of adult respiratory distress syndrome; inflammatory responses; wound healing; reperfusion damage; atherosclerosis; and restenosis following an injury such as balloon angioplasty, atherectomy, and arterial stent placement. The compounds of the present invention may be useful in treating neoplasia and metastasis as well as neurodegenerative diseases, such as Alzheimer""s disease and Parkinson""s disease.
When employed as thrombin or factor Xa inhibitors, the compounds of the present invention may be administered in an effective amount within the dosage range of about 0.1 to about 500 mg/kg, preferably between 0.1 to 10 mg/kg body weight, on a regimen in single or 2-4 divided daily doses.
When employed as inhibitors of thrombin, the compounds of the present invention may be used in combination with thrombolytic agents such as tissue plasminogen activator, streptokinase, and urokinase. Additionally, the compounds of the present invention may be used in combination with other antithrombotic or anticoagulant drugs such as, but not limited to, fibrinogen antagonists and thromboxane receptor antagonists.
Human leucocyte elastase is released by polymorphonuclear leukocytes at sites of inflammation and thus is a contributing cause for a number of disease states. Compounds of the present invention are expected to have an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, and in the treatment of emphysema. The leucocyte elastase inhibitory properties of compounds of the present invention are determined by the method described below. Cathepsin G has also been implicated in the disease states of arthritis, gout and emphysema, and in addition, glomerulonephritis and lung infestations caused by infections in the lung. In their end-use application the enzyme inhibitory properties of the compounds of Formula I is readily ascertained by standard biochemical techniques that are well-known in the art.
The Cathepsin G inhibitory properties of compounds within the scope of the present invention are determined by the following method. A preparation of partially purified human Cathepsin G is obtained by the procedure of Baugh et al., Biochemistry 15:836 (1979). Leukocyte granules are a major source for the preparation of leukocyte elastase and cathepsin G (chymotrypsin-like activity). Leukocytes are lysed and granules are isolated. The leukocyte granules are extracted with 0.20 M sodium acetate, pH 4.0, and extracts are dialyzed against 0.05 M Tris buffer, pH 8.0 containing 0.05 M NaCl overnight at 4xc2x0 C. A protein fraction precipitates during dialysis and is isolated by centrifugation. This fraction contains most of the chymotrypsin-like activity of leukocyte granules. Specific substrates are prepared for each enzyme, namely N-Suc-Ala-Ala-Pro-Val-p-nitroanilide and Suc-Ala-Ala-Pro-Phe-p-nitroanilide. The latter is not hydrolyzed by leukocyte elastase. Enzyme preparations are assayed in 2.00 mL of 0.10 M Hepes buffer, pH 7.5, containing 0.50 M NaCl, 10% dimethylsulfoxide and 0.0020 M Suc-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. Hydrolysis of the p-nitroanilide substrate is monitored at 405 nm and at 25xc2x0 C.
Useful dose range for the application of compounds of the present invention as neutrophil elastase inhibitors and as Cathepsin G inhibitors depend upon the nature and severity of the disease state, as determined by the attending diagnostician, with a range of 0.01 to 10 mg/kg body weight, per day, being useful for the aforementioned disease states.
Compounds of the present invention that inhibit urokinase or plasminogen activator are potentially useful in treating excessive cell growth disease state. As such compounds of the present invention may also be useful in the treatment of benign prostatic hypertrophy and prostatic carcinoma, the treatment of psoriasis, and as abortifacients. For their end-use application, the potency and other biochemical parameters of the enzyme inhibiting characteristics of compounds of the present invention are readily ascertained by standard biochemical techniques well known in the art. Actual dose ranges for this application will depend upon the nature and severity of the disease state of the patient or animal to be treated as determined by the attending diagnostician. It is to be expected that a general dose range will be about 0.0 1 to 10 mg per kg per day for an effective therapeutic effect.
Additional uses for compounds of the present invention include analysis of commercial reagent enzymes for active site concentration. For example, chymotrypsin is supplied as a standard reagent for use in clinical quantitation of chymotrypsin activity in pancreatic juices and feces. Such assays are diagnostic for gastrointestinal and pancreatic disorders. Pancreatic elastase is also supplied commercially as a reagent for quantitation of xcex11-antitrypsin inplasma. Plasma xcex11-antitrypsin increases in concentration during the course of several inflammatory diseases, and xcex11-antitrypsin deficiencies are associated with increased incidence of lung disease. Compounds of the present invention can be used to enhance the accuracy and reproducibility of these assays by titrametric standardization of the commercial elastase supplied as a reagent. See, U.S. Pat. No. 4,499,082.
Protease activity in certain protein extracts during purification of particular proteins is a recurring problem which can complicate and compromise the results of protein isolation procedures. Certain proteases present in such extracts can be inhibited during purification steps by compounds of the present invention, which bind tightly to various proteolytic enzymes.
The pharmaceutical compositions of the invention can be administered to any animal that can experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.
The pharmaceutical compositions of the present invention can be administered by any means that achieve their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, or ocular routes. Alternatively, or concurrently, administration can be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
In addition to the pharmacologically active compounds, the new pharmaceutical preparations can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically.
The pharmaceutical preparations of the present invention are manufactured in a manner that is, itself, known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders, such as, starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents can be added, such as, the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as, sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as, magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings that, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as, acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as, glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules that may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as, fatty oils or liquid paraffin. In addition, stabilizers may be added.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts, alkaline solutions and cyclodextrin inclusion complexes. Especially preferred salts are hydrochloride and acetate salts. One or more modified or unmodified cyclodextrins can be employed to stabilize and increase the water solubility of compounds of the present invention. Useful cyclodextrins for this purpose are disclosed in U.S. Pat. Nos. 4,727,064, 4,764,604, and 5,024,998.
In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
Compounds of Formula I can be labeled with radioactive iodine as described below or by using an exchange reaction. Exchange of hot iodine for cold iodine is well known in the art. Alternatively, a radioiodine labeled compound can be prepared from the corresponding bromo compound via a tributylstannyl intermediate. See, U.S. Pat. No. 5,122,361, herein incorporated by reference.
The present invention also includes compositions which are useful for in vivo imaging of thrombi in a mammal, wherein the compositions are comprised of a compound of Formula I complexed with a radioactive atom.
The present invention also include diagnostic compositions which are useful for in vivo imaging of thrombi in a mammal, comprising a pharmaceutically acceptable carrier and a diagnostically effective amount of compositions derived from the compounds of Formula I.
The xe2x80x9cdiagnostically effective amountxe2x80x9d of the composition required as a dose will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this dose are well known to skilled practitioners in the medial diagnostic arts. Also, the diagnostically effective amount and method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. In any regard, the dose for imaging should be sufficient for detecting the presence of the imaging agent at the site of a thrombus in question. Typically, radiologic imaging will require that the dose provided by the pharmaceutical composition position of the present invention be about 5 to 20 xcexcCi, preferably about 10 xcexcCi. Magnetic resonance imaging will require that the dose provided be about 0.001 to 5 mmole/kg, preferably about 0.005 to 0.5 mmole/kg of a compound of Formula I complexed with paramagnetic atom. In either case, it is known in the art that the actual dose will depend on the location of the thrombus.
xe2x80x9cPharmaceutically acceptable carriersxe2x80x9d for in vivo use are well known in the pharmaceutical art, and are described, for example, in Remington""s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The pharmaceutical compositions of the present invention may be formulated with a pharmaceutically acceptable carrier to provide sterile solutions or suspensions for injectable administration. In particular, injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspensions in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, or the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e.g., liposomes) may be utilized.
The present invention also encompasses diagnostic compositions prepared for storage or administration. These would additionally contain preservatives, stabilizers and dyes. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. At 1449. In addition, antioxidants and suspending agents may be used.
The in vivo imaging methods of the present invention also offer several advantages over previous imaging techniques for the detection or monitoring of the presence, size, regression or increase of a thrombus. In particular, the present invention provides compounds, compositions and diagnostic compositions have been designed to bind extremely tightly to the thrombin associated with a thrombus and thereby reduce xe2x80x9cbackgroundxe2x80x9d due to circulating radioactivity or paramagnetism arising from unbound imaging agent. Furthermore, in vivo imaging by intracoronary injection of the compounds, compositions or diagnostic compositions of the present invention, is expected to be almost instantaneous since these imaging agents would saturate the thrombin bound to the thrombus immediately.
Accordingly, the present invention also includes methods for in vivo imaging of a thrombus in a mammal, comprising the steps of: (1) administering to a mammal a diagnostically acceptable amount of a compound, composition, or diagnostic composition of the present invention and (2) detecting a thrombus in a blood vessel.
The term xe2x80x9cin vivo imagingxe2x80x9d as used herein relates to methods of the detection of a thrombus in a mammal, as well as the monitoring of the size, location and number of thrombi in a mammal, as well as dissolution or growth of the thrombus.
In employing the compounds, compositions or diagnostic compositions in vivo by this method, xe2x80x9cadministeringxe2x80x9d is accomplished parenterally, in either a systemic or local targeted manner. Systemic administration is accomplished by injecting the compounds, compositions by diagnostic compositions of the present invention into a convenient and accessible vein or artery. This includes but is not limited to administration by the ankecubutal vein. Local targeted administration is accomplished by injecting the compounds, compositions or diagnostic compositions of the present invention proximal in flow to a vein or artery suspected to contain thrombi distal to the injection site. This includes but is not limited to direct injection into the coronary arterial vasculature to image coronary thrombi, into the carotid artery to image thrombi in the cerebral vasculature, or into a pedal vein to image deep vein thrombosis of the leg.
The detecting of a thrombus by imaging is made possible by the presence of radioactive atoms localized at such thrombus.
The radioactive atoms associated with the compositions and diagnostic compositions of the present invention are preferably imaged using a radiation detection means capable of detecting gamma radiation, such as a gamma camera or the like. Typically, radiation imaging cameras employ a conversion medium (wherein the high energy gamma ray is absorbed, displacing an electron which emits a photon upon its return to the orbital state), photoelectric detectors arranged in a spatial detection chamber (to determine the position of the emitted photons), and circuitry to analyze the photons detected in the chamber and produce an image.